6-mercaptopurine nucleosides and method of preparing them



United States Patent 3,il741,93tl -MERCAETGEURHNE NUCLEOSEDES AND METHUD9h PREPARHQG THEM George H. Hitchings, Yonkers, and Irving Goodman,

White Plains, N.Y., assignors to Burroughs Wellcome 31 60. (USA) inc,Tuclsahoe, N.Y., a corporation of New York No Drawing. Filed Jan. 5,1959, Ser. No. 784,864 Qlairns priority, application Great liritain Aug.11, W55 22 Claims. (6!. 26tl--211.5)

The present invention relates to 9-glycosido-6-mercaptopurines and to aprocess for preparing the same.

The ability of 6-rnercaptopurine to interfere with cell divisionhasfound a useful expression in the production of remissions of acuteleukemia. The mechanism of action of -rnercaptopurine is believed to beas an antagonist of the natural 6-substituted purines, which exist incellular compositions combined through glycoside linkages.

The biological activity of G-mercaptopurine is modiiied, and becomesmore selective in the new compounds with the result that the range ofusefulness of the growth inhibitor is extended.

The usual method of preparing purine glycosides is by the reaction of aheavy metal salt of the purine with a glycosyl halide. In the presentinstance this method is inoperable presumably because the metal saltsare attached to the sulfur of the mercaptan group. However, it has beenfound that -benzylrnercaptopurine will form a suitable silver salt, andthat debenzylation of the eventual 9-glyccsido--benzylmercaptopurinescan be effected by treating with sodium in liquid ammonia.

In our copending application Serial No. 574,575, now abandoned, we havedescribed a method of preparing nucleosides of 6-marcaptopurines whichconsists of preparing first an acylated (thereby protected) nucleosideof a o-chloropurine and converting that to the acylated nucleoside of a6-mercaptopurine.

The synthesis of nucleosides of 6-chloropurines being inconvenient,though feasible, it is often preferable to start with preformed purinenucleosides available commercially. These are particularly inosine (la)and guanosine (lb). These are first protected by acetylation orbenzoylation of the sugar hydroriyls, and then converted to acylatednucleosides of 6-chloropurines by reaction with phosphorous oxychloridein the presence of a hydrogen halide acceptor. Considering thesensitivity of these nucleosides it is not surprising that thisprocedure is feasible only under special conditions. As the hydrogenhalide-acceptor, weak tertiary organic bases are essential, and dimethyland diethylaniline are preferred. Pyridine, picolines and lutidines canalso be employed, but result in more tarformation.

The chloropurineacylnucleosides is then reacted with a sulfur-bearingreagent. For this purpose thiourea is satisfactory but a salt of athiocarboxylic acid is even better. in both cases it is to be presumedthat unstable sulfurcontaining intermeriates (thio-uronium salts andthiocarboxylates) are formed and these are hydrolyzed repidly under theconditions of the reaction:

The choice of the salt of a thiocarboxylic acid is one EfilldfifibPatented Jan. 22, 1%53 ice dithio-oxalic acid are readily available.Potassium dithiooxalate is probably the most convenient as beinginexpensive and available commercially in pure form.

Finally as in other lines of synthesis, the protective acyl groups areremoved, conveniently by the action of ammonia in alcoholic solution.

The 6-chloropurine acylnucleosides can also be reacted with otherreagents. Of particular interest is the reaction with thiohexoseswhereby are obtained 6,9-diglycosides of mercaptopurine (or ofthioguanine). These compounds (Vii) and their esters (VI) are oftherapeutic value in their own right.

These reactions are illustrated in the chart of reactions withsodium-l-thioglucose leading to the 6-(1'-thioglu cosides) (VI and Vii).It is believed that these particular compounds have the ti-configuration and are 1- thio-fl-d-glucopyranosides. However, neitherthe pyranose form nor the B-configuration have yet been proved beyondquestion. Therefore, at this point, we refrain from writing the stericform of this portion of these compounds.

Eight g. of chloromercuri-6-chloropurine was added to a solution of 7 g.l-chloro-Z,3,5-triacetylribofuranose in 100 ml. anhydrous xylene. Thesolution was refluxed for 48 hours, filtered and the filtrate wasconcentrated to a syrup. The product was an impure oil.

EXAMPLE 2 9-Rz'bofuranosyl-d-Mercaptopurine Va) Two g. of the syrup(III) was refluxed with 0.2 g. thiourea and 0.2. g. sodium acetate inabsolute ethanol for 18 hours. The resulting solution of the thiouroniumsalt TV was filtered, treated with H 8 to remove any remaining Hg,decolorized with Darco, concentrated to a syrup and taken up in absoluteethanol. The ethanolic solution was saturated with NH; and left for 18hours at room temperature. A very small amount (50 mg.)

of product was obtained.

A max.=225, 321 at pH 1; 234, 311 at pH 11; the product gives a positiveMohlisch test and was shown by paper chromatography to have a mobilityresembling that of the corresponding gluccsyl derivative.

EXAMPLE 3 6 -H yzfroxy -9- (2, 3,5 -Trl' -O-A cetylribofuranosyl)-Purine (II ac) 15 g. of inosine (I) is suspended in 200 ml. pyridine.To the suspension is added 100 ml. acetic anhydride. After stirringuntil homogeneous, the mixture is kept on the steam bath for 2 hours.The resulting clear ambercolored solution is concentrated to semi-solidstate in vacuo. 250 ml. of ice Water is added to the residue, whereuponthe solid dissolves with the evolution of heat. Upon cooling for minutesin an ice bath the white crystalline product appears. After keeping at 4C. for one hour, the crystals are collected by filtration and washedwith ice water. Yield, 19.5 g.; (89% of theory). M.P. 230. A max.=245 mu(in absolute EtOH).

EXAMPLE 4 6-C1ti0r0-9- (2,3,5-Tri-O-A cetylribofuranosyl) -Purz'ne(Illac) 16.0 g. of triacetylinosine (H) is intimately mixed with 10 m1.of dimethylaniline. To the mixture is added 100 ml. of POCI Thesuspension is heated on the steam bath for 4 hours (anhydrousconditions). The homogeneous dark brown solution is then concentrated invacuo on the steam bath to remove the excess POCl The remaining brownsyrup is dissolved in 250 ml. CHCl The CHCl solution is washed 5 timeswith 75 ml. portions of H 0 in a separatory funnel, after which thewater washings are at pH 5 to 6. The CHCl layer is dried over anhydrousNa SO and is concentrated to a syrup.

.Yield, 800 mg. (62% of theory).

v 6 chloro 9 (2,3,5-tri (Hlczc).

- ethanol saturated centratedin vacuo to about 5 bath. 40 mg. of pure6-MP Mp. at pH 11.

- and kept at room temperature for 18 Final traces of solvent areremoved under high vacuum. The yield of crude product isolated as asyrup is 14.8 g. (89% of theory). A max.=265 m (in H O).

EXAMPLE 5 6-]l 1'ercapl'0-9- 2,3, 5 -Tri-O-A celylribofw'anosyl) -Purilze (IVac) 1.3 g. of (lilac) is dissolved in 10 ml. of absolute ethanol.To this solution is added a solution containing 0.6 g. of sodiumthioacetate in 10 ml. of absolute ethanol. The clear solution wasrefluxed on the steam bath for 1 hour. The Warm solution was filtered,and upon cooling the product crystallized in the form of white plates.max.=325 my in absolute ethanol.

EXAMPLE 6 6 -M ercapzo-9- 2,3,5 -Tri -O-A cetylrib 0 furanosyl -Purine(IVac) In ten ml. of absolute ethanol was dissolved 2.5 g. of Oacetylribofuranosyl)-purine To this was added a suspension of 1 g. ofpotassium dithiooxalate (COSK) in 25 m1. of absolute ethanol. Themixture was heated at reflux for 1 hour. It was then filtered and thefiltrate was concentrated in EXAMPLE 7 6-Merdapt0-9-Ribofuranosylpurine(Va) mg. of (IV) was dissolved in 25 ml. of absolute with NH;,. Thesolution was kept at room temperature for 18 hours after which it wasconml. and cooled in an ice riboside (V) was isolated (60% of theory). Amax.=225, 321 .at pH 1; 234, 311

EXAMPLE 8 d-Glucothiopyrahosyl-Q( 2,3,5 -Tri-O-A cetylribofurnosyl)-Purine Vlac) 13 g. of crystalline product (VIac) was isolated (66% oftheory). A max.=280

mm (in absolute ethanol).

EXAMPLE 9 6-Glzzc0thiopyranos yl-9-Riboyuranosylpurine VIIa) 5 g. of(VIac) was dissolved in '70 ml. ethanolic NH hours. The solution wasconcentrated in vacuo to 10 ml. Upon adding 10 ml. anhydrous ether, theproduct crystallized. Yield, 2.8 g. (72% of theory). max.=280 m (in HO).

EXAMPLE 10 6-H ydr0xy-9- (2,3,5 -Tri-0-Benzoylribofuranosyl Purine (Had)Five g. of inosine was suspended in The mixture was cooled to 0 in coldsuspension, 25 ml. of CHCI benzoyl chloride was added. The mixture washeated at for 2 hours, allowing the CHCl to distill on.

a The resultant syrup was dissolved in 100 ml. of CHCI 10 ml. ofpyridine. an ice bath. To the followed by 10 g. of

derivative. Yield, 10 g. (92% of theory) N=9.61%. Theory N=9.68%. xmax.=230 and 265 my in alcohol.

EXAMPLE l1 6-Chl0i'o-9-(2,3,5-Tri-0-Benzoylribofuranosyl) -Pz4rine(Iliad) Five g. of tribenzoylinosine (Example 10) was suspended in 5 ml.of dimethylaniline. To this suspension was added 61 g. of POCl whereuponthe solid dissolved. The mixture was heated at 100 for 4 hours. Theresulting light yellow solutionwas poured into 800 ml. of ice water-CHCl(1:1 by volume). The water layer was extracted twice with Cl-ICl TheCHCl layer was washed twice with saturated NaHCO solution and then withwater. After drying over Na SO the CHCL, layer was concentrated in vacuoto a syrup. The resulting syrup was dissolved in 100 ml. of absolutealcohol and poured into 600 ml. of water. Upon acidification to pH 2with HCl, a precipitate was formed. Yield 6 g. of crude product. Amax.=230 and 260 m in alcohol. The 230 mu peak is characteristic of thebenzoyl derivatives while the 260 peak is similar to that of the parentpurine, 6-chloropurine with a peak at 265 (aqueous solution, pH 1). Theproduct contains gives a positive Mohlisch test.

EXAMPLE 12 6-5 1 ercapt-9- (2 ,3 ,5 -Tri-O-B enzoy lrib 0 furzmosylPurine (IVad) EXAMPLE l36-6lucotltiopyranosyl-9-(2,3,5-Tri-Olie-rlz0yZribofuranosyl) -Purine (V1ad) of the chloronucleoside of Example 11 was dissolved in 50 ml. ofabsolute ethanol. To this solution was added a solution containing 1 g.of sodium thioglucose dihydrate in ml. of water. The mixture was heatedat 100 for 15 minutes, then filtered. From the fiiterate was isolated0.8 g. of crude product with max.=228 and 280 me in 50% ethanol. The 280mp.

Cl (organic) an Two g.

eak is characteristic of the 6-purinethoiglycosides. Upon hydrolysisthis yields G-rnercaptopurine. The prodnot still contains the benzoylgroups as shown by the 228 mp peak.

EXAMPLE l4 Fifty g. of guanosine (commercial grade) was suspended in 500ml. of pyridine. T 0 this was added 250 ml. of acetic anhydride. Themixture was heated at 100 for 17 hours. The resulting solution waspoured into 1600 ml. of water and extracted with CH'Cl (3- 200 ml.portions). The CHCl solution was washed twice with saturated NaHSOsolution (2 x 200 ml.), twice with 200 ml. portions of H 0, dried overNa SO and concentrated to a syrup in vacuo. The syrup, on triturationwith anhydrous ether formed a crude solid product (63 g.). A max.=:258mp. at pH 6', 265 m at EXAMlLE l5 Twenty-five g. of the above ester wassuspended in 35 ml. of dimethylaniline. To this was added 240 g. of POClThe mixture was heated at under reflux for 4 hours. The resultingsolution was concentrated in vacuo on the steam bath to a syrup. Thesyrup was dissolved in 200 ml. of CHCl which solution was extractedseveral times with 75 ml. portions of H 0 until the pH of the washingswas about 4. After drying over Na S0 the CHCl layer was concentrated invacuo to a syrup. To this was added anhydrous ether. The resultinghygroscopic solid was dried in vacuo. Yield 12 -g. A max. 252 and 285 m,in alcohol. Contains chlorine (organic). Positive Mohlisch test.

EXAMPLE l6 Z-Amino-d-Mermp t0-9- (2 ,3 ,5 Tri-GAcetylrib ofurzmosyl)-Pztrine (ll be) One g. of the above chloro compound was dissolved in 25ml. of absolute alcohol. To this was added a solution of 0.5 g. ofsodium thioacetate in 5 ml. of absolute alcohol. The mixture wasrefluxed for 2 hours, filtered, and concentrated to dryness in vacuo.The solid residue was treated with 50 ml. of water. A precipitate formedwith U.V. characteristics similar to the parent purine compound,thioguanine. U.V., )t max. at 250 and 340 m in absolute alcohol. The 340peak is characteristic of the mercapto group of thioguanine. A positiveMohlisch test indicates the presence intact of the ribosyl group.

*XAMPLE l7 Z-Amino-d-Mercapto-fi-D rib0;furanosylpurine (l Vb) One-tenthg. of Z-aminoG-mercapto-9-triacetylribofuranosylpurine (Example 16) wasdissolved in 2.5 cc. of alcohol saturated with ammonia. The solution wasallowed to stand over-night at room temperature and was then evaporatedin vacuo. The residue, after washing with absolute ethanol was a brownamorphous powder. it gave a positive ylohlisch test and had the ultraviolet absorption of a thioguanine derivative. (r max. (H 0 260 and 34-0my" At pH ll, 7\ max. 250 and 320 m The peaks at 340 and 320 m are verydistinct and characteristic.)

EXAMPLE 1% Tribenzoyl Guanosine (Hbd) Benzoylation was accomplished bythe method described for the benzoylation of inosine (Example 10). Theproduct was isolated as a crystalline solid, Ml. 205-8". N=ll.36%.Calculated N=ll.75%. A :230, 270 in alcohol.

EXAMPLE 19 2-Amin0-6-Ch l0;'09(2,3 ,5 -Tri-O-Bez .z0y iri '00 fumnosyl)Farina (Hl'bd) 2Amin0-6-Gluc0thiopymnosyl-9-(2,3,5-Tri-O-Benzoylribofumnosyl)-Pzzriue (Vlbd) One-half g. of the d-chloro compoundof Example 19 was treated with 0.25 g. of thioglucose dihydrate by theo-chloropuriue and reacting method of Examples 3 and 13. as a whitecrystalline solid. t max.=230 and 300 mu. Upon hydrolysis, thethioglucosyl linkage is split leaving a product,2-amino-6-mercapto-9tribenzoylribofuranosylpurine, with A max. 230 and340 at pH 1 and 230 and 312 at pH 11. The 230 peak indicates that thebenzoyl ribose portion of the molecule is intact.

What we claim is:

1. The method of preparing nucleosides selectedv from the classconsisting of nucleosides of G-mercaptopurine,2-arnino-6-mercaptopurine, purine-6-thioglycoside and 2-aminopurine-o-thioglycoside which comprises converting consisting ofinosine and guanosine to its O-acyl derivative, reacting the acylatednucleoside with phosphorus oxychloride in the presence of a hydrogenhalide acceptor, so as to form the acylated nucleoside of thecorresponding 6-chloropurine, reacting the latter with a reagent selectefrom the class consisting of thiourea, the alkali thiohexoses and thesalts of aliphatic thiocarhoxylic acids and deacylating the product withalcoholic ammonia to yield the desired nucleoside.

, 2. The method of preparing nucleosides selected from the classconsisting of nucleosides of side and 2-aminopurine-G-thioglycosidewhich comprises The product was isolated the product with alcoholicammonia.

3. The method of preparing nucleosides selected from the classconsisting of nucleosides of 6-mercaptopurine and2-amino-6-mercaptopurine which comprises converthydrogen halideacceptor, so as to form the acylated nucleoside of the corresponding6-chloropurine, reacting the latter compound with thiourea anddcacylating the product with alcoholic ammonia.

4. The method of preparing nucleosides selected from the classconsisting of nucleosides of 6-mercaptopurine and2-amino-6-mercaptopurine which com rises convertlug a reagent of theclass consisting of inosine and guanoderivative, reacting the acylatednucleoammonia.

5. The method of preparing nucleosides selected from the classconsisting of nucleosides of 6-mercaptopurine andZ-amino-6-mercaptopurine which comprises converting a reagent of theclass consisting of inosine and guanosiue to its O-acyl derivative,reacting the acylated nucleoside with phosphorus oxychloride in thepresence of a hydrogen halide acceptor, so as to form the acylatednucleoside of the corresponding 6-ch1oropurine, reacting the lattercompound with an alkali salt and deacylating the product with alcoholicammonia.

6. The method of preparing an O-acylated nucleoside which comprisesconverting a reagent of the class consisting of inosine and guanosine toits O-acyl derivative, reacting the acylated nucleoside with phosphorusoxychloride in the presence of a hydrogen halide acceptor, so as to formthe acylated nucleoside of the corresponding the latter compound with analkali thiohexose.

7. The method or" preparing an O-acylated nucleoside which comprisesconverting a reagent of the class consisting of inosine and guanosine toits O-acyl derivative, reacting the acylated nucleoside with phosphorusoxychloride in the presence of a hydrogen halide acceptor, so as .toform the acylated nuclcoside of the corresponding 6- of dithiooxalicacid I sisting of inosine and chloropurine and reacting the lattercompound with thiourea.

8. The method of preparing an O-acylated nucleoside which comprisesconverting a reagent of the class consisting of inosine and guanosine toits G-acyl derivative, reacting the acylated nucleoside with phosphorusoxychloride in the presence of a hydrogen halide acceptor, so as to formthe acylated nucleoside of the corresponding 6- chloropurine andreacting the latter compound with an alakali salt of thioacetic acid.

9. The methodjof preparing an O-acylated nucleoside which comprisesconverting a reagent of the class conguanosine to its O-acyl derivative,reacting the acylated nucleoside with phosphorus oxychloride in thepresence of a hydrogen halide acceptor, so as to form the acylatednucleoside of the corresponding 6-chloropurine and reacting the lattercompound with an alkali salt of dithiooxalic acid.

10. The method of preparing nucleosides selected from the classconsisting of nucleosides of 6-thioglycosido purine and2-amino-6-thioglycosido purine which comprises converting a reagent ofthe class consisting of O-triacylinosine and O-tn'acylguanosine into its6-chloro derivative by reaction With phosphorus oxychloride in thepresence of a hydrogen halide acceptor, reacting the 6-chloro compoundwith an alkali thiohexose to form the 6-thi0- glycoside and deacylatingthis product with alcoholic ammonia.

11. The method of preparing nucleosides selected from the classconsisting of nucleosides of o-mercaptopurine and2-amino-6-mercaptopurine which comprises converting a compound of theclass consisting of O-triacylinosine and O-triacylguanosine' to its6-chloro derivative by reaction with phosphorus oxychloride in thepresence of a hydrogen halide acceptor, reacting the 6-chl0ro compoundWith thiourea to give the 6-mercapto derivative and deacylating thelatter with alcoholic ammonia.

12. The method of preparing nucleosides selected from the classconsisting of nucleosides of G-mercaptopurine and2-amino-6-mercaptopurine which comprises convertcapto derivative anddeacylating the latter with alcoholic ammonia.

13. The method of preparing nucleosides selected from the classconsisting of nucleosides of 6-mercaptopurine and2-amino-6-mercaptopurine which comprises converting a compound of theclass consisting of O-triacylinosine and O-triacylguanosine to its6-chloro derivative by resides of 2 -amino-6-chloropurine with an aceticacid to alkali salt of-thiogive the 6-mercapto derivative anddeacylating the latter with alcoholic ammonia.

16. The method of preparing nucleosides selected from the classconsisting of nucleosides of G-mercaptopurine and2-amino-6-mercaptopurine which comprises reacting 18.Purine-9-fi-d-ribcfuranoside-6-thioglucoside. a compound selected fromthe class consisting of the tY'll9. Purine-96-d-triaeetylriboside-6-thioglucoside. acylnucleosides of 6-chloropurineand the triacylnucleo- 29. 2-aminopurine B t-cl-ribofutancside-6-thioglucosides of 2-amino-6-cl1loropurine with analkali salt of (11- side. thiooxalic acid to give the G-mercaptoderivative and 5 21. 2-aminopurine 9 p-d-triacetylribofuranosicle-6-deacylating the latter with alcoholic ammonia. tnloglucoside.

17. A compound represented by the formula: 22. 2amino-6-chloropurine-9-fl-cl-triacetylribofurano- Sl-glucose side.

\1, A/ m Reierences Cited in the tile of this patent l X L UNlTED STATESPATENTS I N 2,746,961 Hitchings et a1 May 22, 1956 R 2,852,505 BakerSept. 16, 1958 q RCPCHQ 2,852,596 Goldman et a1 cept. 16, 1958 H G 7OTHER REFERENCES Friedkin: Biochem ct Biophys Am 18, pp. 447-448 63(1955). (Cited in Chem. Abstracts 40, 3516, 1956.)

. Eohnson, In, tal.: lAm/hemSo .78,3863 1956 wherein R is selected fromthe class conslstmg of CH CO, 20 e b c C H CO and hydrogen and R isselected from the class consisting of the amino group and hydrogen.

1. THE METHOD OF PREPRING NUCLEOSIDES SELECTED FROM THE CLASS CONSISTINGOF NUCLEOSIDES OF 6-MERCAPTOPURINE, 2-AMINO-6-MERCAPTOPURINE,PURINE-6-THIOGLYCOSIDE AND 2AMINOPURINE-6-THIOGLYCOSIDE WHICH COMPRISESCONVERTING A REAGENT OF THE CLASS CONSISTING OF INOSINE AND GUANOSINE TOITS O-ACYL DERIVATIVE, REACTING THE ACYLATED NUCLEOSIDE WITH PHOSPHORUSOXYCHLORIDE IN THE PRESENCE OF A HYDROGEN HALIDE ACCEPTOR, SO AS TO FORMTHE ACYLATED NUCLEOSIDE OF THE CORRESPONDING 6-CHLOROPURINE, REACTINGTHE LATTER WITH A REAGENT SELECTED FROM THE CLASS CONSISTING OFTHIOUREA, THE ALKALI THIOHEXOSES AND THE SALTS OF ALIPHATICTHIOCARBOXYLIC ACIDS AND DEACYLATING THE PRODUCT WITH
 17. A COMPOUNDREPRESENTED BY THE FORMULA: